title: ENR_WTmusc_m1355
description: Protocols: Organoids (see growth protocol) wells were first disrupted by strong pipetting and pelleted before lysis. Colon tissue was used for muscle isolation. After flushing with cold PBS, colons were opened in longitudinal and dissected under a bench scope. Using a coverslip, mucosal tissue was completely removed carefully under the dissection scope and muscle was cut in 2-4 cm pieces, wash with sterile PBS twice, spin down and collected in P24 well plates. Pieces of muscle were inspected under the microscope for residual crypts or fat and further cleaned. Pieces were then incubated in DMEM-F12 for 24h before collecting the supernatant (muscle-SN). Muscle-SN was filtered (0,20um) and used directly + ENR factors in organoids cultures (right after splitting). Organoids were exposed to muscle-SN for 5 days. Muscle pieces were fixed in paraformaldehyde (PFA) 4% and processed in paraffin for H&E. Tissues were inspected to confirm the absence of mucosa. Small intestinal crypts were isolated and cultured following previously published protocol (Sato 2013). Organoids were cultured in basal crypt culture medium (BCM) consisting of advanced Dulbecco's modified Eagle medium -F12 supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM Glutamax, 1x N2 [ThermoFisher Scientific 100X, 17502048], 1x B-27 [ThermoFisher Scientific 50X, 17504044], and 1x N-acetyl-L-cysteine [Sigma, A7250]) and overlaid with ENR factors containing 50 ng/ml of murine EGF [Thermo Fisher Scientific, PMG8041], 20% R-Spondin-CM (conditioned medium), kind gift from Calvin Kuo, Stanford University School of Medicine, Stanford, CA, USA), and 10% Noggin-CM. Medium was renewed every other day. For passaging, organoids cultures were washed, and matrigel and organoids were disrupted mechanically by strong pipetting, centrifuged at 200g, 5 min at 4 °C and resuspended in matrigel to plate in P24 wells. References Sato and Clevers. 2013. Primary Mouse Small Intestinal Epithelial Cell Cultures. Epithelial Cell Culture Protocols: Second Edition Organoids were cultured alone or exposed to muscle supernatant (see dissection protocol) for 5 days. Wells contained ENR control medium, ER (without Noggin factor), ENR + muscle supernatant (muscle-SN) or ER + muscle supernatant. RNA isolation was performed following manufacturer instructions (Direct-zol™ RNA MiniPrep, BioSite R-2052). RNA was quantified by spectrophotometry (ND1000 Spectrophotometer, NanoDrop, Thermo Scientific) and 25 ul at a 50 ng/ul concentration of RNA were used for RNA seq. Library preparation and sequencing for organoid RNA seq was performed by Novogene (UK) Co. NEB Next® Ultra™ RNA Library Prep Kit was used to generate the library.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-05-01T00:23:34Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-06-03T20:02:38Z
attributes.tag: External Id
attributes.value: SAMEA6872423
attributes.tag: INSDC center name
attributes.value: Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
attributes.tag: INSDC first public
attributes.value: 2021-05-01T00:23:34Z
attributes.tag: INSDC last update
attributes.value: 2020-06-03T20:02:38Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9181:ENR_WTmusc_m1355
attributes.tag: age
attributes.value: 12 to 15
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: epithelial cell of small intestine
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: organoid culture (ENR medium)
attributes.tag: individual
attributes.value: C
attributes.tag: organism part
attributes.value: epithelium of small intestine
attributes.tag: sample name
attributes.value: E-MTAB-9181:ENR_WTmusc_m1355
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: stimulus
attributes.value: intestinal smooth muscle culture supernatant
attributes.tag: strain
attributes.value: C57BL/6